GZMK+CD8+ T cells Target A Specific Acinar Cell Type in Sjögren’s Disease

Sjögren’s Disease (SjD) is a systemic autoimmune disease without a clear etiology or effective therapy. Utilizing unbiased single-cell and spatial transcriptomics to analyze human minor salivary glands in health and disease we developed a comprehensive understanding of the cellular landscape of healthy salivary glands and how that landscape changes in SjD patients. We identified novel seromucous acinar cell types and identified a population of PRR4+CST3+WFDC2− seromucous acinar cells that are particularly targeted in SjD. Notably, GZMK+CD8 T cells, enriched in SjD, exhibited a cytotoxic phenotype and were physically associated with immune-engaged epithelial cells in disease. These findings shed light on the immune response’s impact on transitioning acinar cells with high levels of secretion and explain the loss of this specific cell population in SjD. This study explores the complex interplay of varied cell types in the salivary glands and their role in the pathology of Sjögren’s Disease.


Supplemental Figures:
Figure S1.(a) Barplot of changes in cell proportion in disease and SSA positivity across all cell types.(b) Dotplot of genes found to be specific to Leiden clusters and used to annotate those clusters.(c) Dotplots of differentially expressed genes in Sjögren's and the cell types those genes are differentially expressed in.(d) Dotplots of differentially expressed genes in SSA+/-and the cell types those genes are differentially expressed in.(e) Dotplots of genes differentially expressed across Sjögren's and anti-SSA positivity and the cell types those genes are differentially expressed in.      Figure S8.(a) Representative marker genes spatial plots for seromucous (MUC7) and mucous (MUC5B) acini, fibroblasts (LUM), and T cells (CD3E).Note SjD accompanies major shifts in seromucous acinar cell (SMAC) cell composition, T cells, and fibrosis.(b) Cartoon depicting cellular colocalization and cooccurrence analyses.Reference cell types were integrated from existing scRNA-seq (Figure 1e) data using Cell2Location to infer cell type locations in spRNA-seq.(c) Autocorrelations of cell types form a colocalization matrix that reconstructs the usual architecture of the glands.In SjD, the general architecture remains preserved; however, shifts in colocalizations can be appreciated in multiple cell types.(d) Shown are CD8+ exhausted T cells and PRR4+CST3+WFDC2-seromucous acinar cells (SMACs) along with manually annotated ducts and immune infiltrates.Top tissue is from a healthy volunteer and bottom tissue is from a patient with a diagnosis for Sjögren's.

Figure
Figure S2.(a) Distribution of marker genes in SjD and non-SjD ISH samples.Samples are downsampled so that an equal number of cells are taken from all samples.(b) UMAP of the cells from ISH.There is a clear division by patient diagnosis.(c) UMAP colored by ISH quantification.

Figure
Figure S3.(a) UMAP colored by Gaussian mixture model (GMM) cluster.The sample number of clusters were used as input channels from the ISH data.(b) Distribution of ISH quantification for each cluster of cells.(c)Samples with cells colored by GMM cluster.GMM clusters broadly correspond to acinar, interstitial or immune tissue from one of three groups of patients: patients with a high focus score, patients with a low focus score or no focus score and antibody positivity, or patients who were antibody negative and had no focus score.Two non-SjD patients were outliers and clustered uniquely.

Figure S4 .
Figure S4.Transforming scRNA-seq data from gene expression to indices (a) reveals new organizations of cells and clinical feature-specific clusters.(b) UMAP of cells after transformation to an index S-score space with coloring by a new application of the Leiden clustering algorithm.(c) Proportions of index clusters that represent annotations in the original single-cell space.Some clusters are close to a single cell type; others contain a variety of cell types.There are no one-to-one mappings between index clusters and original annotations.(d) UMAP on index S-scores colored by original annotation.Some clusters are enriched for clinical features and multiple regions are homogenous in their clinical phenotype.(e) The original scRNA-seq UMAP overlaid with the index clusters.The two most specific index clusters are both found in the T cell island in the UMAP as well as the clusters associated with antigen-presenting cells.(f) Ratios of Sjögren's to non-Sjögren's and anti-SSA positive to negative patients in each of the index clusters.Two index clusters are found at the extremes of the plot: one with high SjD specificity and the other with high anti-SSA positive specificity.These index clusters are composed of T cells, B cells, dendritic cells, and macrophages.

Figure S5 .
Figure S5.Interferon response increased across cell types with SSA positivity.(a) Cells in the index UMAP colored by patient's diagnosis and SSA positivity.(b) Changes in index expression with disease diagnosis and SSA positivity.(c) Feature plots of IFN score in SSA+ and SSA-patients.Except in M2 macrophages and endothelial populations, IFN response score is low across cell types in SSA negative individuals.(d) Barplot of IFN response S-score across cell types in SSA positive and negative patients.IFN response increases in every cell population.

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Figure S6.PhenoCycler-Fusion 35-plex spatial proteomics confirms changes in glandular composition and cellular interactions.(a) Dot plot of multiple protein expression of cellular phenotypes (left-side of dot plot; EPCAM to CD20) and cellular state markers (right side of dot plot; HHP3 to IFNG).(b) Cellular proportion changes in SjD compared with nonSjD.(c) Per-cell log2 fold-changes in protein expression in SjD compared with non-SjD were calculated for all phenotype and segmented cells.(d) Cellular neighborhoods in SjD compared with non-SjD.(e) CellChat ligand-receptor analysis corroborating neighborhood analysis and the importance of B-and T-lymphocyte co-stimulatory signaling in SjD.(f) Representative PhenoCycler-Fusion immunofluorescent images from a lymphocytic focus showing infiltration with T-and B-lymphocytes around ducts and acini.

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Figure S7.(a) Evaluation of the induction of apoptosis after transfection of recombinant GZMK, GZMB, β-Gal, or Naive (mock) in immortalized NS-SV-TTAC Acinar Cells using Annexin V/PI staining and flow cytometric analysis.100,000 events were measured and performed in biological duplicate (except for -Gal) *-p<0.05,ANOVA.(b) Optimization of protein transfection with GZMK in HSG cells.Granzyme K levels were normalized to -Actin and determined ratios are compared to control.(c) Transfection of GZMK and GZMB into NS-SV-TTAC exhibits time-dependent degradation over 48 hours.Granzyme K and B levels were normalized to -Actin and determined ratios are compared to naive Sample.(d) GZMK transfection induced dose-dependent increases in pIRF3 by Western immunoblot in pSGEC.(e) THP1-macrophages were transfected with proteins (i.e., GZMK, GZMB, GFP, and mock) and analyzed by fluorescence microscopy showing GZMK induces cytosolic relocalization of mtDNA to the cytosol.Image is representative of the findings from three biological replicates.(f) Engineered cell lines were used to measure IRF Pathway and Type I IFN induction using THP1-ISH Lucia and HEK-IFNa/b SEAP cells, respectively.Assays were performed on technical triplicates from biological duplicates and compared using ANOVA; results presented are a single representative experiment from the three experimental replicates.

Table 1 :
List of antibodies and dyes used for flow cytometry of minor salivary glands.

Table 2 .
List of PhenoCycler-Fusion Validated Antibodies used for 35-plex Spatial Phenotyping